The present invention relates generally to cytofectin and adjuvant compositions. More particularly, the present invention provides compositions useful as cytofectins and as adjuvants, as well as methods for facilitating the transfection of nucleic acids into cells and for enhancing the humoral immune response of vertebrates to polynucleotide-based vaccines.
Cytofectins are used to enhance the delivery of biologically active agents, particularly polynucleotides, proteins, peptides, and drug molecules, by facilitating transmembrane transport or by encouraging adhesion to biological surfaces. Some bioactive substances do not need to enter cells to exert their biological effect, because they operate either by acting on cell surfaces through cell surface receptors or to cell surfaces by interacting with extracellular components. However, many natural biological molecules and their analogues, including proteins and polynucleotides, or foreign substances, such as drugs, which are capable of influencing cell function at the subcellular or molecular level are preferably incorporated within the cell in order to produce their effect. For these agents, the cell membrane presents an impermeable selective barrier.
Successful intracellular delivery of agents not naturally taken up by cells has been achieved by exploiting the natural process of intracellular membrane fusion, or by direct access of the cell""s natural transport mechanisms, which include endocytosis and pinocytosis (Duzgunes, N., Subcellular Biochemistry 11:195-286 (1985)). In addition, the cell membrane barrier can be overcome by complexing the agent to be delivered or transfected with lipid formulations closely resembling the lipid composition of natural cell membranes. These lipids are able to fuse with the cell membranes on contact, and in the process, the agents associated with the lipid complexes or aggregates are delivered intracellularly. Lipid aggregates comprising charged lipids can not only facilitate intracellular transfers by fusing with cell membranes but also by overcoming charge repulsions between the cell membrane and the agent to be delivered.
Cellular delivery of beneficial or interesting proteins can be achieved by introducing expressible DNA or mRNA into cells, a technique known as transfection. Nucleotide sequences introduced in this way can produce the corresponding protein encoded by the nucleotide sequence. The therapy of many diseases could be enhanced by the induced intracellular production of peptides which could remain inside the target cell, be secreted into the local environment of the target cell, or be secreted into the systemic circulation to produce their effect. Various techniques for introducing the DNA or mRNA precursors of bioactive peptides into cells include the use of viral vectors, including recombinant vectors and retroviruses, which have the inherent ability to penetrate cell membranes. However, the use of such viral agents to integrate exogenous DNA into the chromosomal material of the cell carries a risk of damage to the genome and the possibility of inducing malignant transformation. Another aspect of this approach which restricts its use in vivo is that the integration of DNA into the genome accomplished by these methods implies a loss of control over the expression of the peptide it codes for, so that transitory therapy is difficult to achieve and potential unwanted side effects of the treatment could be difficult or impossible to reverse or halt.
A major advance in the area of DNA transfection was the discovery that certain synthetic cationic lipids, such as DOTMA, in the form of liposomes or small vesicles, could interact spontaneously with DNA to form lipid-DNA complexes that are capable of fusing with the negatively charged lipids of the cell membranes, resulting in both uptake and expression of the DNA (see, e.g., Feigner, P. L. et al., Proc Natl Acad Sci USA 84:7413-7417 (1987) and U.S. Pat. No. 4,897,355, the disclosures of which are incorporated herein by reference). The well-known Lipofectin(trademark) reagent (Bethesda Research Laboratories, Gaithersburg, Md.), an effective agent for the delivery of highly anionic polynucleotides into living tissue culture cells, comprises positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. In part, the effectiveness of cationic lipids as cytofectins is thought to result from their enhanced affinity for cells, many of which bear regions of high negative charge on their membrane surfaces. Also in part, the presence of positive charges on a lipid aggregate comprising a cationic lipid enables the aggregate to bind polyanions, especially nucleic acids. Lipid aggregates prepared in this way can spontaneously attach to negative charges on cell surfaces, can fuse with the plasma membrane, and can efficiently deliver functional polynucleotides into cells. More recently, other cationic lipids, including diesters and diethers of modified Rosenthal Inhibitor (RI) compounds, have been found to be effective cytofectin compounds (see, e.g., U.S. Pat. Nos. 5,459,127 and 5,264,618, the disclosures of which are incorporated herein by reference).
In the late 1980s, it was discovered that direct intramuscular (i.m.) injection of lipid-DNA complexes results in measurable protein expression, and also that xe2x80x9cnakedxe2x80x9d plasmid DNA (pDNA) can be taken up and expressed in muscle to a greater extent than lipid-DNA complexes (Felgner, 1997)). One of the first applications of pDNA injection technology was the induction of an immune response. In 1991, it was first reported that mice could be immunized against HIV gp120 by i.m. vaccination with gp120 plasmid DNA (Felgner et al., 1991), and that mice could be protected from a lethal challenge of influenza virus after DNA immunization with influenza nucleoprotein (NP) antigen. Protection obtained after immunization with the highly conserved NP antigen extended across two different viral strains (Ulmer et al., 1996)). Numerous publications in the field of polynucleotide-based vaccination followed thereafter (Boyer et al., 1996; Boyer et al., 1997; Davis et al., 1997; Wang et al., 1997; Agadjanyan et al., 1998; Heppell et al., 1998; Lodmell et al., 1998; Vanderzanden et al., 1998)).
A problem often encountered in the course of polynucleotide-based vaccination is insufficient or suboptimal humoral response. To obtain a stronger humoral and/or cellular response, it is common to administer such vaccines in an immunogenic composition containing an adjuvant, a material which enhances the immune response of the patient to the vaccine. Adjuvants are useful generally for improving the immune response of an organism to a particular immunogen and are commonly included in vaccine compositions to increase the amount of antibodies produced and/or to reduce the quantity of immunogen and the frequency of administration.
A variety of adjuvants have been reported to effect differing levels of immune response enhancement to polynucleotide-based vaccination. Examples of such adjuvant materials include semi-synthetic bacterial cell wall-derived mono-phosphoryl lipid A (Sasaki, S. et al., Infection and Immunity 65(9):3250-3258 (1997)), small molecule immunostimulators (Sasaki, S. et al., Clin Exp Immunol 111:30-35 (1998)), and saponins (Sasaki, S. et al., J Virol 72(6):4391-4939 (1998)). The immune response from i.m. pDNA vaccination has also been enhanced through the use of cationic lipids (e.g., Ishii, N. et al., Aids Res Hum Retroviruses 13(16):1421-1428 (1997)), Okada, E. et al., J Immunology 159:3638-3647 (1997); Yokoyama, M. et al., FEMS Immunol Med Microbiol 14:221-230 (1996); Gregoriadis, G. et al., FEBS Letters 402:107-110 (1997); Gramzinski, R. A. et al., Molecular Medicine 4:109-118 (1998); Klavinskis, L. S. et al., Vaccine 15(8):818-820 (1997); Klavinskis, L. S. et al., J Immunology 162:254-262 (1999); Etchart, N. et al, J Gen Virology 78:1577-1580 (1997); Norman, J. et al., in Methods in Molecular Medicine, Vol. 9; DNA Vaccines: Methods and Protocols, D. B. Lowrie and R. Whalen, eds., Chapter 16, pp. 1-13 (1999)). Cationic lipids were originally studied as cytofectins to enhance the transfection and delivery of pDNA into cells in vitro (see, e.g., cytofectins disclosed and claimed in U.S. Pat. Nos. 5,334,761, 5,459,127 and 5,264,618, the disclosures of which are incorporated herein by reference); however, further development has led to successful specific applications of protein delivery in vivo (Wheeler, C. J. et al., Proc Natl Acad Sci USA 93:11454-11459 (1996); Stephan, D. J. et al, Human Gene Therapy 7:1803-1812 (1996); DeBruyne, L. A. et al., Gene Therapy 5:1079-1087 (1998)).
The present invention is directed in certain embodiments to cytofectin and adjuvant compositions, as well as to immunogenic compositions comprising immunogens and such adjuvant compositions. In other embodiments, the present invention relates to methods for facilitating the transfection and delivery of bioactive agents into cells, and to methods for enhancing the humoral immune responses of vertebrates to polynucleotide-based vaccines.
The present invention also provides methods for facilitating transfection and delivery of bioactive agents into cells, as well as methods for enhancing the immune response of vertebrates to immunization, particularly pDNA immunization. The present invention is especially useful in this latter regard by providing enhanced humoral immune response, as evidenced by the level of antibody titers, to polynucleotide-based vaccines. Elevation of antibody levels is particularly advantageous in applications where antibody levels from the immunogen-encoding nucleotide sequence alone are sub-optimal. In a related advantage, if the desired level of antibodies is produced with a given dose of pDNA, the amount of pDNA necessary to reach the predetermined antibody titer level can be reached using a lower pDNA dose. For pDNA vaccination applications, this advantage is important because acceptable vaccination volumes, coupled with functional limits on the concentration of pDNA, define an upper limit on a given vaccine dose. This advantage is particularly important for vaccines containing multiple plasmids, each of which must be present in sufficient quantity to elicit an immune response to its particular transgene.
With regard to the polynucleotide-based vaccination aspect, the methods of the present invention are useful prophylactically to protect a vertebrate from a disease, therapeutically to treat a diseased vertebrate, or both. In certain preferred embodiments, the present invention is directed to a method for immunizing a vertebrate by administering to the vertebrate an immunogenic composition which includes a nucleotide sequence that encodes an immunogen, and an adjuvant composition comprising a novel cationic lipid compound having two quaternary ammonium headgroups bridged by a linker. The immunogen-encoding nucleotide sequence, upon incorporation into the cells of the vertebrate, produces an immunologically effective amount of an immunogen (e.g., an immunogenic protein). The adjuvant composition of the present invention enhances the immune response of the vertebrate to the immunogen.
In certain embodiments, the compositions of the present invention further include one or more co-lipids or other lipid aggregate-forming components such as, for example, phospholipids, lysophospholipids, lysolipids and cholesterol. Such co-lipids include cationic, anionic and neutral lipids. Other appropriate co-lipids for use in the present invention are known to or may be determined by those skilled in the art.
One aspect of the present invention is a composition comprising a novel cationic lipid compound having hydrophobic tails and two quaternary ammonium headgroups bridged by a linker, the cationic lipid compound having a structure according to general formula (I) or (II) described below.
In certain preferred embodiments, where the cationic lipid compound has a structure according to formula (I), the linker is optionally substituted C1 to C10 alkyl or alkyloxy, or optionally substituted C1 to C10 alkenyl or alkenyloxy. In a particularly preferred embodiment, the cationic lipid compound is PentaEG-bis-DMRIE, the chemical structure of which is shown in FIG. 1B. The chemical name of PentaEG-bis-DMRIE is penta(ethylene glycol), xcex1, xcfx89-bis-(xc2x1)-N-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide ether.
In certain other embodiments, the linker preferably includes a ureyl linkage (i.e., xe2x80x94NRxe2x80x94C(O)xe2x80x94NRxe2x80x94, where R is H or optionally substituted C1 to C10 alkyl or alkenyl), a bis-ureyl linkage (i.e., xe2x80x94NRxe2x80x94C(O)xe2x80x94NRxe2x80x94Rxe2x80x2xe2x80x94NRxe2x80x94C(O)xe2x80x94NRxe2x80x94, where R is H or optionally substituted C1 to C10 alkyl or alkenyl, and Rxe2x80x2 is optionally substituted C1 to C10 alkyl or alkenyl), or a peptide linkage (i.e., xe2x80x94C(O)xe2x80x94NRxe2x80x94, where R is H or optionally substituted C1 to C10 alkyl or alkenyl). These compositions may further include one or more co-lipids.
In certain other preferred embodiments, where the cationic lipid compound has a structure according to formula (I) described below, the linker has DNA and/or cell receptor binding affinity. Such linkers may enhance the effectiveness of the lipid in interacting with nucleotides and/or cell membranes. Examples of moieties having such binding affinity include, for example, amino acids, peptides, saccharides, polypeptides, polysaccharides, proteins, polyamines, peptidomimetic moieties and histories. Specific examples of polyamines having such binding affinity include spermine, spermidine, and derivatives thereof. In particularly preferred embodiments incorporating a peptide moiety, the cationic lipid is HB-DMRIE-Ox-Trp-xcex3-DMRIE or PEG34-bis-But-DMRIE-propylamide, the chemical structures of which are shown in FIG. 1B. The chemical name of HB-DMRIE-Ox-Trp-xcex3-DMRIE is (xc2x1)-N-[4-(Nxe2x80x2-(3xe2x80x2-tryptophanylaminopropyl))-Nxe2x80x2,Nxe2x80x2-dimethyl-2xe2x80x2, 3xe2x80x2-bis(tetradecyloxy)-1xe2x80x2-propanaminiumyl]-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide. The molecular formula of HB-DMRIE-Ox-Trp-xcex3-DMRIE is C84H161Br2N5O6. The chemical name of PEG34-bis-But-DMRIE-propylamide is poly(ethylene glycol)-34 bis-[(xc2x1)-N-(Nxe2x80x2-propylbutyramido)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide].
In yet other preferred embodiments, where the cationic lipid compound has a structure according to formula (II), the linker bridging the quaternary ammonium headgroups includes a bis-ureyl linkage. In certain embodiments, the cationic lipid compound is a dimer, wherein the hydrophobic lipid tails, represented by groups R1 to R4, are identical. In particularly preferred embodiments, the cationic lipid compound is SBDU-DMRIE, SBGU-DMREE or SHGU-DMRIE, the chemical structures of which are provided in FIG. 1A. Another common name of SBDU-DMRIE is butane bis-DU-DMRIE. The chemical name of SBDU-DMRIE is 1,4-bis-(Nxe2x80x2-butyl-(4-(N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium))-ureyl-butane. The molecular formula is C80H166O6N6. Another common name of SHGU-DMRIE is hexane bis-1,6-GU-DMRIE. The chemical name of SHGU-DMRIE is 1,4-bis-(Nxe2x80x2-propyl-(4-(N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium))-ureyl-hexane. The molecular formula is C80H166O6N6. Another common name of SBGU-DMRIE is butane bis-GU-DMRIE. The chemical name of SBGU-DMRIE is 1,4-bis-(Nxe2x80x2-propyl-(4-(N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium))-ureyl-butane. The molecular formula is C80H166O6N6. These compositions may also optionally include one or more co-lipids.
Another aspect of the present invention is an immunogenic composition comprising an immunogen and an adjuvant composition comprising a cationic lipid compound according to general formula (I) or (II) described below. Preferably, the immunogen is provided by an immunogen-encoding nucleotide sequence which, most preferably, is a plasmid DNA, or a portion thereof. The immunogenic composition may further include one or more co-lipids.
Still another aspect of the present invention is a method for inducing an immune response in a vertebrate by administering to the vertebrate an immunogenic composition, which includes one or more immunogen-encoding nucleotide sequences, and an adjuvant composition which includes one or more cationic lipid compounds according to general formula (I) or (II) described below, in an amount sufficient to generate an immune response to the encoded immunogen. The vertebrate is preferably a mammal and, most preferably, is a human.
Yet another aspect of the present invention is a method useful for delivering a biologically active agent to a cell of a plant or animal. The method involves preparing a lipid aggregate comprising the biologically active agent and a composition including one or more cationic lipid compounds according to general formula (I) or (II) described below, followed by contacting the cell with the lipid aggregate. This method is useful for both in vivo and in vitro delivery to cells, and may be utilized for the transfection of cells.
Yet another aspect of the present invention is a pharmaceutical preparation comprising a cytofectin/adjuvant composition including a cationic lipid compound according to general formula (I) or (II) together with a pharmacologically effective amount of a therapeutic agent. The cytofectin composition facilitates the cellular delivery of the therapeutic agent. Preferably, the therapeutic agent is a polynucleotide, such as an antisense RNA or DNA molecule. The polynucleotide can encode an immunogen, a natural hormone, or a synthetic analogue of a natural hormone, or it can encode a gene product that is deficient or absent in a disease state, and administration of the gene product to a vertebrate has a therapeutic effect.